Studies of nucleosome positioning in vitro and in vivo have revealed the importance of sequence specific histone-DNA interactions and nuclease sensitive regions acting as boundaries in determining positioning. Positioning has been defined for nucleosome containing 5S genes from two species, the SV-40 enhancer and several yeast genes. We have begun study of ternary complexes formed from histones, defined sequence DNA and the eukaryotic regulatory factor, TFIIIA. Methods have been developed which allow isolation of yeast plasmids as chromatin. Characterization of the chromatin has been done for the propositus plasmid. A novel method for isolation of nucleoprotein complexes is being employed to facilitate purification of episomal chromatin. We have characterized the chromatin structure of other, regulated yeast genes and now will isolate these as plasmid constituents to determine the proteins required for their chromosomal organization. Studies of transcriptionally regulated genes (sea urchin histones and human c-myc) have revealed alterations in chromatin structure which correlate with the capacity of the gene to be transcribed. Using genetic manipulations, we have defined the region of the c-myc gene which is necessary for its tissue specific transcription. We are now attempting to isolate regulatory proteins which interact with the control loci for these two gene sets. A segment of a collagen gene from sea urchin has been isolated and sequenced. Developmental studies indicate that the gene is transcribed at blastula stage and thereafter.